In order to understand the development and function of the thymus, both in terms of T cell differentiation and stromal cell environmental support, we have undertaken a molecular approach to identify genes that are uniquely expressed in this organ. We have created an RT-PCR based subtracted cDNA library from fetal thymic stromal cells. The novel cDNAs were then screened for expression in various tissues and in a set of SV40 transformed thymic epithelial cell lines. Multiple full length cDNA clones were isolated from a SCID thymus library. The three novel genes identified were named Epithin, Spatial, and Thymic Stroma Co-Transporter (TSCOT). We are currently focusing on experiments with two of these gene products (TSCOT and Epithin). 1). The lab has now established multiple mouse strains which show thymus-specific expression patterns of targeted exogenous genes (EGFP and LacZ) in different thymic epithelial cell compartments using both transgenic and knock-in approaches. A transgenic mouse line containing 3.1kb of the TSCOT promoter driving EGFP was established to identify the thymic stromal cell lineage in which TSCOT is expressed. Surprisingly, this mouse showed EGFP expression in the subcapsular and medullary compartments of the thymus in contrast to the cortex where the endogenous gene is expressed. In another model, a mouse was generated by knocking in the LacZ gene at the TSCOT locus. We showed that LacZ is expressed only in the thymic epithelial cell compartment. Interestingly, expression of these targeted proteins changes during development. Using this information, we are now generating more mouse lines that can be used for specific cell ablation in either a constitutive or inducible fashion in different thymic stromal cell compartments. 1) During the past year, using our established mouse models, in addition to previously available mutant mice, our investigation focused on mechanistic aspects of the process of cortical / medullary compartmentalization. We also studied differentiation at the cellular level using surface marker profiles. In addition, using a targeted neo-self protein in thymic epithelial cells, we were able to observe highly efficient tolerance induction by thymic epithelium containing a limited number of specificially-targeted, neoantigen-expressing cells (several thousand per thymus). The numbers of molecules expressed in them (several thousand per cell) was also low. Finally, we observed that this tolerance induction was only partially affected by deficiency of the Autoimmune Regulatory gene (AIRE). 2) While we were searching for new cell surface markers to define thymic epithelial cells, we found that MHCII and TSCOT were good for identifying such subpopulations. During development of thymic epithelium, expression profiles of these markers by flow cytometry and confocal microscopy revealed that UEA-1+ medullary epithelial cells express two different levels of MHCII while cortical epithelial cells express only one homogenously high level. TSCOT positive cells were mostly CDR1 or 6C3 positive in younger animals, but some became negative for this marker in older age. It is interesting to note that TSCOT+ cells were only a minor subpopulation in thymic epithelial cell preparations (usually less than 10% of total epithelial cells). 3) We were successful in detecting reporter activity in the thymic epithelium of both a 3.1 Kb and a 4.4 Kb TSCOT-CRE transgenic mouse. We also localized EGFP expression in a transgenic mouse expressing nitroreductase under the control of either a 9.1 or a 3.1 Kb TSCOT promoter. All these mice are now being bred to expand their numbers for experiments. 4) TSCOT appears to be an interesting marker for thymic epithelial cell differentiation. Its expression was detected at embryonic day 11, as well as in the nude thymic rudiment, suggesting that it is expressed even before the FoxN1 (nude) gene in thymic differentiation. Furthermore, when the 3.1 and 4.4 Kb TSCOT-CRE mice were crossed with ROSA-Stop-LacZ mice, the lacZ reporter activity was detected in both cortical and medullary epithelial compartments. These results raise the possibility that TSCOT could be a marker for precursor thymic epithelial cells. 5). In collaboraion with Dr. D. Park, we have been successful in identifying proteins involved in interactions with Epithin using the Yeast 2 hybrid technology. In addition, we are continuing our search for the general nature of Epithin's function and the mechanism of its processing during cellular signaling. One of the proteins isolated is an actin-binding protein Filamin, which behaves as a molecular bridge linking the extracellular membrane protein Epithin to the key intracellular cytoskeletal protein actin. When Filamin links Epithin into cortical actin in the cell, Epithin is concentrated in the cell contact sites and a protease activity is then transactivated, resulting in the shedding of the short form of the Epithin protein. Using MALDI-TOF technology with extracts generated from the thymic epithelial cell line 427, we were able to identify the angiogenin1 receptor, Tie2, as a binding protein for epithin. Biochemical analysis is being applied in transfection experiments to verify the type of interaction in both epithelial and endothelial cells.